JPWO2002051998A1 - New protease - Google Patents

Abstract

A novel polypeptide, a polynucleotide encoding the polypeptide, an expression vector comprising the polynucleotide, a cell transfected with the expression vector, an antibody or a fragment thereof binding to the polypeptide, and a process for producing the polypeptide are disclosed. <??>The polypeptide is a novel protease.

Description

Technical field
The present invention relates to novel proteases.
Background art
ADAMTS (A Disintegrin and Metalloprotease with Thrombospondin motif) is a group of molecules including a disintegrin-like domain, a metalloproteinase-like domain, and a thrombospondin type I repeat sequence (hereinafter referred to as TSP-1 repeat sequence). Reported nine human ADAMTS molecules.
Among the human ADAMTS molecules, in ADAMTS4 (aggrecanase-1) and ADAMTS11 (aggrecanase-2), the extracellular matrix aggrecan is located between the 373rd glutamic acid residue and the 374th alanine residue (Glu³⁷³-Ala³⁷⁴), Indicating the possibility of being a main enzyme in the degradation of cartilage extracellular matrix aggrecan in arthritis and osteoarthritis (Tortorella MD et al., Science). And Abbaszade {I. et al., J. Biol. Chem., 274, 23443-23450, 1999). ADAMTS2 (procollagen I N-proteinase) is an enzyme that cleaves and removes the N-terminal part of pro-type I collagen, is involved in the conversion of type I collagen from pro-form to mature form, and is important for collagen fiber formation. And a link between abnormalities of the gene and VIH type Ehlers-Danlos syndrome has been shown (Collig A. et al., Am. J. Hum. Genet., 65, 308-). 317, 1999).
That is, it has been shown that ADAMTS molecules are involved in metabolism such as degradation and maturation of extracellular matrices such as aggrecan and collagen.
On the other hand, chronic renal failure is a disease characterized by glomerular sclerosis and renal interstitial fibrosis, and qualitative changes and / or quantitative increases in extracellular matrix components are regarded as major pathogenesis and progression mechanisms. . In an experiment using a renal insufficiency disease model, gene transfer of decorin, a protein that specifically suppresses the action of transforming growth factor β (TGF-β) (Isaka, Y. et al., Nature, Med., 2, 418-423, 1996) ) And anti-TGF-β administration (Ziyadeh FN et al., Proc. Natl. Acad. Sci. USA, 97, 8015-8020, 2000; Sharma @ K. et al., Diabetes, 45, 522-530. And Border WA et al., Nature, 346, 371-374, 1990) show that inhibiting or inhibiting the physiological function of TGF-β leads to the treatment of chronic renal failure. It is believed that.
However, under the current situation in which there is no satisfactory therapeutic agent for chronic renal failure, despite being expected, no TGF-β inhibitor has been put on the market to date.
Disclosure of the invention
Since TGF-β is a differentiation and growth factor exhibiting a wide variety of physiological functions, inhibiting all of the physiological functions of TGF-β is necessary from the viewpoint of side effects in the treatment of chronic renal failure in which long-term administration is assumed. It is a danger. It is desirable to suppress or inhibit only the part related to the qualitative change and quantitative increase of the extracellular matrix component in the physiological action of TGF-β.
An object of the present invention is to provide a novel protease, which is induced by TGF-β and is involved in extracellular matrix metabolism, which is useful as a screening tool for a therapeutic agent for chronic renal failure, and a novel polynucleotide encoding the same. It is in.
The present inventors have conducted intensive studies in order to solve the above-mentioned problems. As a result, the human fetal kidney cDNA was composed of a sequence consisting of amino acids 1 to 1224 in the sequence represented by SEQ ID NO: 2, A polynucleotide encoding a novel protease consisting of 10 amino acid residues was found. Furthermore, the present inventors have found that the N-terminal partial fragment consisting of 750 amino acid residues of this novel protease also has sufficient protease activity. In addition, the protease is considered to be (1) a protease involved in the metabolism of extracellular matrix because it is classified as an ADAMTS protease, and (2) that it is actually expressed in human kidney. (3) Expression was induced by TGF-β in primary cultured kidney cells, and (4) the gene expression level was increased in renal failure model animals. Thus, the protease of the present invention is a polypeptide that causes renal failure. By screening for a substance that inhibits its protease activity using the polypeptide of the present invention, the physiological activity of TGF-β The present inventors have clarified that it is possible to screen a substance useful as a therapeutic agent for chronic renal failure, which suppresses or inhibits only a portion related to qualitative change and quantitative increase of extracellular matrix components, and completed the present invention.
That is, the present invention
[1] (1) a sequence consisting of amino acids 1 to 750 in the amino acid sequence represented by SEQ ID NO: 2, (2) a sequence consisting of amino acids 1 to 750 in the amino acid sequence represented by SEQ ID NO: 2 In the sequence consisting of amino acids, an amino acid sequence in which 1 to 10 amino acids have been deleted, substituted, and / or inserted, or (3) an amino acid sequence represented by SEQ ID NO: 1 A polypeptide comprising an amino acid sequence in which 1 to 10 amino acids have been deleted, substituted, and / or inserted in a sequence consisting of amino acids, and which has protease activity;
[2] a polypeptide comprising a sequence consisting of the 1st to 750th amino acids in the amino acid sequence represented by SEQ ID NO: 2 and further exhibiting protease activity;
[3] (1) an amino acid sequence in which 1 to 10 amino acids have been deleted, substituted, and / or inserted in the sequence consisting of amino acids 1 to 750 in the amino acid sequence represented by SEQ ID NO: 2 Or (2) an amino acid sequence in which 1 to 10 amino acids have been deleted, substituted, and / or inserted in the sequence consisting of amino acids 1 to 1224 in the amino acid sequence represented by SEQ ID NO: 2 A polypeptide comprising protease activity;
[4] (1) homology with the sequence consisting of amino acids 1 to 750 in the amino acid sequence represented by SEQ ID NO: 2, or (2) the first homology in the amino acid sequence represented by SEQ ID NO: 2 A polypeptide comprising an amino acid sequence having a homology of 90% or more with a sequence consisting of the amino acids Nos. 1224 and showing protease activity;
[5] (1) homology with the sequence consisting of amino acids 1 to 750 in the amino acid sequence represented by SEQ ID NO: 2, or (2) the first homology in the amino acid sequence represented by SEQ ID NO: 2 A polypeptide comprising an amino acid sequence having 90% or more homology with the sequence consisting of the amino acids Nos. 1224; and exhibiting protease activity;
[6] (1) consisting of the 1st to 750th amino acids in the amino acid sequence represented by SEQ ID NO: 2, or (3) the 1st to 1224th amino acids in the amino acid sequence represented by SEQ ID NO: 2 A polypeptide consisting of:
[7] a polynucleotide encoding the polypeptide of [1] to [6];
[8] an expression vector comprising the polynucleotide of [7];
[9] a cell transfected with the expression vector according to [8];
[10] an antibody or a fragment thereof that binds to the polypeptide of [1] to [6];
[11] The method for producing the polypeptide according to [1] to [6], comprising culturing the cell according to [9] and recovering the polypeptide according to [1] to [6];
[12] (1) The polypeptide according to [1] to [6], and (2) α.₂Contacting macroglobulin with (3) a test compound, and
The polypeptide and α₂-A step of analyzing whether or not macroglobulin forms a complex that is not dissociated by SDS and / or a reducing agent
A method for detecting whether a test compound inhibits the protease activity of said polypeptide;
[13] a detection step according to the method of [12], and
Step of selecting a substance that inhibits protease activity
A method for screening for a substance that inhibits the protease activity of the polypeptide according to [1] to [6];
[14] A method for screening a substance for treating chronic renal failure by the method according to [13];
[15] a detection step according to the method of [12], and
Formulation process
A method for producing a pharmaceutical composition for treating chronic renal failure, comprising:
About.
As used herein, “protease activity” refers to α, a protease inhibitory protein present in serum.₂-Means a property capable of forming a complex with macroglobulin which does not dissociate with sodium dodecyl sulfate (SDS) and / or a reducing agent.
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described in detail.
[1] The polypeptide of the present invention
In the polypeptide of the present invention,
(1) a polypeptide comprising a sequence consisting of amino acids 1 to 750 in the amino acid sequence represented by SEQ ID NO: 2;
(2) In one or more positions of the sequence consisting of the 1st to 750th amino acids in the amino acid sequence represented by SEQ ID NO: 2, 1 to 10 amino acids as a whole are deleted, substituted, and / or A polypeptide comprising the inserted amino acid sequence and exhibiting protease activity (hereinafter referred to as a functionally equivalent variant);
(3) Homology with the sequence consisting of the 1st to 750th amino acids in the amino acid sequence represented by SEQ ID NO: 2, or 1st to 1224th in the amino acid sequence represented by SEQ ID NO: 2 A polypeptide containing an amino acid sequence having a homology of 90% or more with an amino acid sequence and exhibiting protease activity (hereinafter, referred to as a homologous polypeptide)
Is included.
The polypeptide of the present invention, “a polypeptide comprising a sequence consisting of the first to 750th amino acids in the amino acid sequence represented by SEQ ID NO: 2” is the first polypeptide in the amino acid sequence represented by SEQ ID NO: 2. The polypeptide is not particularly limited as long as it contains a sequence consisting of the amino acids 750 to 750 and exhibits protease activity.
(1a) a polypeptide consisting of the 1st to 750th amino acids in the amino acid sequence represented by SEQ ID NO: 2;
(1b) having an amino acid sequence in which an appropriate marker sequence or the like has been added to the N-terminal and / or C-terminal of the sequence consisting of amino acids 1 to 750 in the amino acid sequence represented by SEQ ID NO: 2, And a fusion polypeptide exhibiting protease activity;
(1c) The amino acid sequence represented by SEQ ID NO: 2 at the C-terminus of the sequence consisting of the 1st to 750th amino acids, from the 751th to 1224th amino acids in the amino acid sequence represented by SEQ ID NO: 2 Or an amino acid sequence to which a sequence in which 1 to 473 amino acids have been deleted from the C-terminus has been added [that is, the C-terminus is any of amino acids 751 to 1224; The amino acid sequence represented by No. 2 or its C-terminal deletion sequence "];
(1d) an amino acid sequence represented by SEQ ID NO: 2 or a C-terminal deletion sequence thereof, which has an amino acid sequence to which an appropriate marker sequence or the like is further added at its N-terminal and / or C-terminal; Active fusion polypeptides
And so on.
In the present specification, a method for determining whether or not a certain polypeptide (hereinafter, referred to as a test polypeptide) exhibits “protease activity” (hereinafter, may be referred to as a “method for determining protease activity”) is as follows. "Α, a protease inhibitory protein present in serum₂-Is not particularly limited as long as it can be determined whether or not it shows "a property capable of forming a complex with macroglobulin that is not dissociated by SDS and / or a reducing agent". The test polypeptide and α, a protease inhibitory protein present in serum₂By contacting with macroglobulin and analyzing whether or not it forms a complex that does not dissociate with SDS and / or a reducing agent [eg, 2-mercaptoethanol (2-ME)]; Specifically, for example, it can be confirmed by the method described in Example 4.
α₂-Macroglobulin is a protease inhibitory protein present in serum and is known to form a complex with many proteases. The formation of this complex is dependent on the protease activity, and the complex formed₂-It is known that it is not dissociated by SDS or a reducing agent (for example, 2-ME) because it is an amide bond with macroglobulin (Feinman RD, et al., Ann. New York Acad. Sci., And Kuno {K. et al., J. Biol. Chem., 274, 18821-18826, 1999).
The polypeptide (1a), that is, the “polypeptide consisting of the 1st to 750th amino acids in the amino acid sequence represented by SEQ ID NO: 2” is a novel polypeptide consisting of 750 amino acid residues exhibiting protease activity. It is a protease. The polypeptide (1a) corresponds to a partial polypeptide of “a polypeptide consisting of the 1st to 1224th amino acids in the amino acid sequence represented by SEQ ID NO: 2”.
As the marker sequence in the polypeptide of the present invention, for example, a sequence for easily confirming expression of the polypeptide, confirming intracellular localization, or purification and the like can be used. For example, a FLAG tag, Examples include a hexa-histidine tag, a hemagglutinin tag, or a myc epitope.
The functional equivalent variant of the present invention comprises 1 to 10, preferably 1 to 7, at one or more positions in the sequence consisting of amino acids 1 to 750 in the amino acid sequence represented by SEQ ID NO: 2. , More preferably 1 to 5 amino acids (for example, 1 to several amino acids), as long as the polypeptide contains a deleted, substituted, and / or inserted amino acid sequence and exhibits protease activity. And its origin is not limited to humans.
For example, it includes not only a human variant of the polypeptide consisting of amino acids 1 to 750 in the amino acid sequence represented by SEQ ID NO: 2, but also a non-human organism (eg, mouse, rat, hamster, Or canine-derived functional equivalent variants. Furthermore, based on a polynucleotide encoding those natural polypeptides (ie, a human-derived mutant or a functionally equivalent variant derived from a non-human organism), or represented by SEQ ID NO: 2 Polypeptides produced using polynucleotides that have been artificially modified by genetic engineering based on polynucleotides encoding polypeptides consisting of amino acids 1 to 750 in the amino acid sequence, and the like are included. As used herein, the term “variant” refers to an individual difference between identical polypeptides in the same species, or a difference between homologous polypeptides between several species.
Mutants in humans of the polypeptide consisting of amino acids 1 to 750 in the amino acid sequence represented by SEQ ID NO: 2 or functionally equivalent variants derived from organisms other than humans can be obtained by those skilled in the art. Based on information on the nucleotide sequence of a polynucleotide encoding a polypeptide consisting of the 1st to 750th amino acids in the amino acid sequence represented by SEQ ID NO: 2 (for example, the nucleotide sequence represented by SEQ ID NO: 1) , Can be obtained. Unless otherwise specified, the gene recombination technique may be carried out according to a known method (for example, Sambrook, J. et al., "Molecular Cloning-A Laboratory Manual", Cold Spring Harbor Laboratory, NY, 1989). It is possible.
For example, an appropriate primer or probe is designed based on information on the nucleotide sequence of a polynucleotide encoding a polypeptide consisting of amino acids 1 to 750 in the amino acid sequence represented by SEQ ID NO: 2, and the primer Alternatively, a probe and a sample (eg, a total RNA or mRNA fraction, a cDNA library, or a phage library) derived from a target organism [eg, a mammal (eg, human, mouse, rat, hamster, or dog)] And a hybridization method using the polymerase chain reaction (PCR) method (Saiki, RK, et al., Science, 239, 487-491, 1988) to obtain a polynucleotide encoding the polypeptide, Expression of the polynucleotide using an appropriate expression system Was, expressed polypeptide, for example, by the method described in Example 4, by confirming that shows the protease activity can acquire the desired polypeptide.
In addition, the above-mentioned polypeptide which has been artificially modified by genetic engineering can be prepared by a conventional method, for example, site-specific mutagenesis (Mark, DF, et al., Proc. Natl. Acad. Sci. USA, 81, 5662-5666, 1984), and obtains a polynucleotide encoding the polypeptide, and expresses the polynucleotide using an appropriate expression system. The desired polypeptide can be obtained by confirming that it exhibits protease activity by the method described.
Functionally equivalent variants of the present invention include, for example,
(2a) In one or more positions of the sequence consisting of the 1st to 750th amino acids in the amino acid sequence represented by SEQ ID NO: 2, 1 to 10 amino acids as a whole are deleted, substituted, and / or A polypeptide having an inserted amino acid sequence and exhibiting protease activity;
(2b) In one or more positions of the sequence consisting of the 1st to 750th amino acids in the amino acid sequence represented by SEQ ID NO: 2, 1 to 10 amino acids as a whole are deleted, substituted, and / or A fusion polypeptide which has an amino acid sequence inserted and further added with an appropriate marker sequence or the like at the N-terminus and / or C-terminus thereof and further exhibits protease activity;
(2c) In one or a plurality of positions of the sequence consisting of the 1st to 750th amino acids in the amino acid sequence represented by SEQ ID NO: 2, 1 to 10 amino acids as a whole are deleted, substituted, and / or A sequence consisting of amino acids 751 to 1224 in the amino acid sequence represented by SEQ ID NO: 2 or a sequence in which 1 to 473 amino acids have been deleted from the C-terminus has been added to the C-terminal thereof. A polypeptide having the specified amino acid sequence and exhibiting protease activity;
(2d) 1 to 10 amino acids as a whole are deleted, substituted, and / or at one or more positions in the sequence consisting of the 1st to 750th amino acids in the amino acid sequence represented by SEQ ID NO: 2 A sequence consisting of amino acids 751 to 1224 in the amino acid sequence represented by SEQ ID NO: 2 or a sequence in which 1 to 473 amino acids have been deleted from the C-terminus has been added to the C-terminal thereof. A fusion polypeptide having an amino acid sequence to which an appropriate marker sequence or the like is further added at the N-terminus and / or C-terminus thereof, and exhibiting protease activity
And so on.
The homologous polypeptide of the present invention has homology to the sequence consisting of amino acids 1 to 750 in the amino acid sequence represented by SEQ ID NO: 2, or the first amino acid in the amino acid sequence represented by SEQ ID NO: 2. Although it is not particularly limited as long as it contains an amino acid sequence having a homology of 90% or more with the sequence consisting of the amino acids Nos. With respect to the sequence consisting of the 1st to 750th amino acids in the amino acid sequence represented, or the sequence consisting of the 1st to 1224th amino acids in the amino acid sequence represented by SEQ ID NO: 2, preferably 95% or more , More preferably 98% or more, still more preferably 99% or more. As the homologous polypeptide of the present invention, homology with the sequence consisting of amino acids Nos. 1 to 750 in the amino acid sequence represented by SEQ ID NO: 2 or the first amino acid sequence represented by SEQ ID NO: 2 The amino acid sequence has a homology of 90% or more (more preferably 95% or more, further preferably 98% or more, particularly preferably 99% or more) with the sequence consisting of the amino acids Nos. 1224; Polypeptides that exhibit activity are more preferred.
The term “homology” in the present specification refers to a value obtained by BLAST (Basic local allocation search search tool; Altschul, SF et al., J. Mol. Biol., 215, 403-410, 1990). And the homology of the amino acid sequences can be determined using a BLAST search algorithm. Specifically, the bl2seq program of the BLAST package (sgi32-bit version, version 2.0.12; obtained from NCBI) (Tatiana A. Tatusova and Thomas @ L. Madden, FEMS @ Microbiol. Lett., 174, 247-250, 1999) And can be calculated according to the default parameters. The program name “blastp” is used as a pairwise alignment parameter, the Gap insertion Cost value is “0”, the Gap expansion Cost value is “0”, “SEG” is used as a query array filter, and “BLOSUM62” is used as a Matrix. Is used for each.
The polypeptide of the present invention has been described above. Examples of the polypeptide of the present invention include “a polypeptide consisting of the 1st to 750th amino acids in the amino acid sequence represented by SEQ ID NO: 2” and “SEQ ID NO: 2 A polypeptide consisting of the 1st to 1224th amino acids in the amino acid sequence represented by: ", one or more of the sequence consisting of the 1st to 750th amino acids in the amino acid sequence represented by SEQ ID NO: 2 In each part, 1 to 10 (preferably 1 to 7, more preferably 1 to 5) amino acids are composed of an amino acid sequence in which deletion, substitution, insertion, and / or addition has been performed, and Or one or more of the sequence consisting of amino acids 1 to 1224 in the amino acid sequence represented by SEQ ID NO: 2. In each part, 1 to 10 (preferably 1 to 7, more preferably 1 to 5) amino acids are composed of an amino acid sequence in which deletion, substitution, insertion, and / or addition has been performed, and Is preferable, "a polypeptide consisting of the 1st to 750th amino acids in the amino acid sequence represented by SEQ ID NO: 2", or "a polypeptide having the first amino acid sequence represented by SEQ ID NO: 2". ~ A polypeptide consisting of the 1224th amino acid "is more preferred.
[2] The polynucleotide of the present invention
The polynucleotide of the present invention is not particularly limited as long as it is a polynucleotide encoding the polypeptide of the present invention. For example, bases 1 to 2250 in the base sequence represented by SEQ ID NO: 1 And particularly preferably a polynucleotide consisting of the 1st to 2250th bases in the base sequence represented by SEQ ID NO: 1. Note that the term “polynucleotide” in the present specification includes both DNA and RNA.
The method for producing the polynucleotide of the present invention is not particularly limited, and examples thereof include (1) a method using PCR, and (2) a conventional genetic engineering technique (that is, transformation using a cDNA library). A method of selecting a transformant containing a desired cDNA from the transformant), or (3) a chemical synthesis method. Hereinafter, each manufacturing method will be sequentially described.
In the method (1) using PCR, the polynucleotide of the present invention can be produced, for example, by the following procedure.
That is, mRNA is extracted from a human cell or tissue having the ability to produce the polypeptide of the present invention. Next, based on the nucleotide sequence of the polynucleotide encoding the polypeptide of the present invention, a pair of primer sets capable of sandwiching the full length of the mRNA corresponding to the polypeptide of the present invention, or a part of the mRNA set A pair of primer sets capable of sandwiching the region is prepared. By performing a reverse transcriptase-polymerase chain reaction (RT-PCR) using the extracted mRNA as a template, a full-length cDNA of the polypeptide of the present invention or a part thereof can be obtained.
More specifically, first, total RNA including mRNA encoding the polypeptide of the present invention is extracted from cells or tissues capable of producing the polypeptide of the present invention by a known method. Examples of the extraction method include a guanidine thiocyanate hot phenol method, a guanidine thiocyanate-guanidine hydrochloride method, and a guanidine thiocyanate cesium chloride method.The guanidine thiocyanate cesium chloride method may be used. preferable. Cells or tissues capable of producing the polypeptide of the present invention include, for example, a Northern blotting method using a polynucleotide encoding the polypeptide of the present invention or a part thereof, or an antibody specific to the polypeptide of the present invention. Can be specified by a Western blotting method using
Subsequently, the extracted mRNA is purified. mRNA may be purified according to a conventional method. For example, mRNA can be purified by adsorbing it onto an oligo (dT) cellulose column and then eluting it. If desired, mRNA can be further fractionated by sucrose density gradient centrifugation or the like. In addition, even without extracting mRNA, commercially available extracted and purified mRNA can be used.
Next, the purified mRNA is subjected to a reverse transcriptase reaction in the presence of, for example, a random primer, an oligo dT primer, and / or a custom-synthesized primer to synthesize a first-strand cDNA. This synthesis can be performed by a conventional method. Using the obtained first-strand cDNA, PCR is carried out using two types of primers sandwiching the full length or a partial region of the target polynucleotide to amplify the target cDNA. The obtained DNA is fractionated by agarose gel electrophoresis or the like. If desired, the target DNA fragment can be obtained by cutting the DNA with a restriction enzyme or the like and connecting it.
In the method (2) using a conventional genetic engineering technique, the polynucleotide of the present invention can be produced, for example, by the following procedure.
First, a single-stranded cDNA is synthesized using reverse transcriptase with the mRNA prepared by the method using PCR as a template, and then a double-stranded cDNA is synthesized from the single-stranded cDNA. Examples of the method include an S1 nuclease method (Estratidis, A. et al., Cell, 7, 279-288, 1976) and a Land method (Land, H. et al., Nucleic Acids Res., 9, 2251-2266, 1981). , O. The Joon @ Yoo method (Yoo, OJ, et al., Proc. Natl. Acad. Sci. USA, 79, 1049-1053, 1983), or the Okayama-Berg method (Okayama, H. and Berg, P., Mol. Cell. Biol., 2, 161-170, 1982).
Next, after preparing a recombinant plasmid containing the double-stranded cDNA, the recombinant plasmid is introduced into Escherichia coli (for example, DH5α strain, HB101 strain, or JM109 strain) for transformation, for example, against tetracycline, ampicillin, kanamycin, or the like. A recombinant is selected using drug resistance as an index. For example, when the host cell is Escherichia coli, the transformation of the host cell is performed by the method of Hanahan (Hanahan, DJ, Mol. Biol., 166, 557-580, 1983), that is, CaCl 2₂, MgCl₂Alternatively, the method can be carried out by adding the recombinant DNA to competent cells prepared in the presence of RbCl. In addition, as a vector, a phage vector such as a lambda system can be used in addition to the plasmid.
Methods for selecting a transformant having the cDNA of interest from the transformants thus obtained include, for example, (i) a transformant screening method using a synthetic oligonucleotide probe, and (ii) PCR (Iii) a transformant screening method using an antibody against the polypeptide of the present invention, or (iv) a transformant screening method using a selective hybridization translation system. Can be adopted.
In the transformant screening method using the synthetic oligonucleotide probe of the above (i), for example, a transformant having the target cDNA can be selected by the following procedure.
That is, an oligonucleotide corresponding to all or a part of the polypeptide of the present invention is synthesized, and this is probed (³²P or³³(Labeled with P), the DNA of the transformed strain is hybridized with a denatured and fixed nitrocellulose filter or polyamide filter, and the resulting positive strain is searched for and selected. In the case of synthesizing an oligonucleotide for a probe, a nucleotide sequence derived using codon usage can be used, or a plurality of nucleotide sequences obtained by combining possible nucleotide sequences can be used. . In the latter case, the type can be reduced by including inosine.
In the transformant screening method using the probe prepared by PCR of the above (ii), a transformant having the target cDNA can be selected, for example, by the following procedure.
That is, oligonucleotides of sense primers and antisense primers corresponding to a part of the polypeptide of the present invention are synthesized, and PCR is performed by combining them to amplify a DNA fragment encoding all or a part of the target polypeptide. I do. As the template DNA used here, cDNA synthesized by reverse transcription reaction from mRNA of a cell producing the polypeptide of the present invention or genomic DNA can be used. The DNA fragment thus prepared is, for example,³²P or³³A transformant having the target cDNA is selected by labeling with P and performing colony hybridization or plaque hybridization using this as a probe.
In the method for screening a transformant using an antibody against the polypeptide of the present invention (iii), a transformant having the cDNA of interest can be selected, for example, by the following procedure.
That is, in advance, the cDNA is incorporated into an expression vector, the polypeptide is produced on the culture supernatant of the transformant, intracellularly, or on the cell surface, using an antibody against the polypeptide of the present invention and a secondary antibody against the antibody, A desired polypeptide-producing strain is detected, and a transformant having the cDNA of interest is selected.
In the transformant screening method using the selective hybridization / translation system (iv), a transformant having the cDNA of interest can be selected, for example, by the following procedure.
That is, cDNA obtained from the transformed strain is blotted on a nitrocellulose filter or the like, and mRNA separately prepared from cells having the ability to produce the polypeptide of the present invention is hybridized, and then the mRNA bound to the cDNA is dissociated. ,to recover. The recovered mRNA is injected into an appropriate polypeptide translation system, for example, Xenopus oocytes, or translated into a polypeptide using a cell-free system such as rabbit reticulocyte lysate or wheat germ. A transformant having the cDNA of interest is selected by detection using an antibody against the polypeptide of the present invention.
The method of collecting the polynucleotide of the present invention from the obtained transformant of interest is a known method (for example, Sambrook, J. et al., "Molecular Cloning-A Laboratory Manual", Cold Spring Harbor Laboratory, NY, 1989). Can be implemented according to For example, it can be carried out by separating a fraction corresponding to plasmid DNA from cells and cutting out a cDNA region from the obtained plasmid DNA.
In the method using the chemical synthesis method (3), for example, the polynucleotide of the present invention can be manufactured by binding DNA fragments manufactured by the chemical synthesis method. Each DNA can be synthesized using a DNA synthesizer [for example, Oligo 1000M DNA Synthesizer (manufactured by Beckman) or 394 DNA / RNA Synthesizer (manufactured by Applied Biosystems)].
In addition, the polynucleotide of the present invention can be prepared on the basis of information on the polypeptide of the present invention by a conventional method such as the phosphite triester method (Hunkapiller, M. et al., Nature, 10, 105-111, 1984). Alternatively, it can be produced by chemical synthesis of nucleic acids. The codon for the desired amino acid is known per se and may be arbitrarily selected. For example, it can be determined according to a conventional method in consideration of the frequency of codon usage of the host to be used (Crantham, R. et al., Nucleic Acids Res., 9, r43-r74, 1981). Further, partial modification of the codons of these nucleotide sequences can be performed by a site-specific mutagenesis method using a primer consisting of a synthetic oligonucleotide encoding the desired modification (Mark \ DF) according to a conventional method. Natl. Acad. Sci. USA, 81, 5662-5666, 1984) and the like.
Sequencing of DNA obtained by the various methods described so far can be performed, for example, by the Maxam-Gilbert chemical modification method (Maxam, AM and Gilbert, W., "Methods in Enzymology", 65, 499-559, 1980) and the dideoxynucleotide chain termination method (Messing, J. and Vieira, J., Gene, 19, 269-276, 1982).
[3] Expression vector and cell of the present invention
Eukaryotic or prokaryotic host cells can be transfected by reincorporating the isolated polynucleotide of the present invention into an appropriate vector DNA. In addition, by introducing an appropriate promoter and a sequence related to expression into these vectors, the polynucleotide can be expressed in each host cell.
The expression vector of the present invention is not particularly limited as long as it contains the polynucleotide of the present invention.For example, the polynucleotide of the present invention may be inserted into a known expression vector appropriately selected depending on the host cell used. Can be mentioned.
The cells of the present invention are also not particularly limited as long as they are transfected with the expression vector of the present invention and contain the polynucleotide of the present invention.For example, the polynucleotide of the present invention is transferred to the chromosome of a host cell. It can be an integrated cell or a cell containing an expression vector containing a polynucleotide according to the present invention. In addition, the cells may be cells expressing the polypeptide of the present invention, or cells not expressing the polypeptide of the present invention. The cell of the present invention can be obtained, for example, by transfecting a desired host cell with the expression vector of the present invention.
For example, eukaryotic host cells include cells such as vertebrates, insects, and yeast, and vertebrate cells include, for example, monkey COS cells (Gluzman, Y., Cell, 23, 175-182, 1981), a dihydrofolate reductase-deficient strain of Chinese hamster ovary cells (CHO) (Urlaub, G. and Chasin, LA, Proc. Natl. Acad. Sci. USA, 77, 4216-4220, 1980), human embryonic kidney-derived HEK293 cells, and 293-EBNA cells (Invitrogen) obtained by introducing the EBNA-1 gene of Epstein-Barr virus into the HEK293 cells.
As a vertebrate cell expression vector, those having a promoter, an RNA splice site, a polyadenylation site, and a transcription termination sequence which are usually located upstream of the gene to be expressed can be used. It can have a replication origin. Examples of the expression vector include pSV2dhfr having an early promoter of SV40 (Subramani, S. et al., Mol. Cell. Biol., 1, 854-864, 1981) and pEF-BOS having a human elongation factor promoter. (Mizushima, S. and Nagata, S., Nucleic Acids Res., 18, 5322, 1990), or pCEP4 (Invitrogen) having a cytomegalovirus promoter.
When 293-EBNA cells are used as host cells, pCEP4 (Invitrogen) having an Epstein-Barr virus origin of replication and capable of self-propagation in 293-EBNA cells can be used as an expression vector.
When a COS cell is used as a host cell, it has an SV40 origin of replication as an expression vector, is capable of autonomous growth in COS cells, and further has a transcription promoter, a transcription termination signal, and an RNA splice site. For example, pME18S (Maruyama, K. and Takebe, Y., Med. Immunol., 20, 27-32, 1990), pEF-BOS (Mizushima, S. and Nagata, S., Nucleic) can be used. Acids @ Res., 18, 5322, 1990) or pCDM8 (Seed, B., Nature, 329, 840-842, 1987).
The expression vector is prepared, for example, by a DEAE-dextran method (Luthman, H. and Magnusson, G., Nucleic Acids Res., 11, 1295-1308, 1983), a calcium phosphate-DNA coprecipitation method (Graham, FL. van der Ed.AJ, Virology, 52, 456-457, 1973), commercially available transfection reagents (eg, FuGENE)^TM6 Transfection Reagent; manufactured by Boeringer Mannheim, or an electric pulse perforation method (Neumann, E. et al., EMBO J., 1, 841-845, 1982), etc., can be taken into COS cells. .
Further, when CHO cells are used as host cells, a vector capable of expressing a neo gene functioning as a G418 resistance marker together with an expression vector containing a polynucleotide encoding the polypeptide of the present invention, for example, pRSVneo (Sambrook) J. et al., "Molecular Cloning-A Laboratory Manual", Cold Spring Harbor Laboratory, NY, 1989) or pSV2-neo (Southern, PJ and Berg, P., J. Mol. Appl. 327-341, 1982) and selecting G418-resistant colonies to obtain transfections that stably produce the polypeptide of the present invention. Cells can be obtained.
The cells of the present invention can be cultured according to a conventional method (for example, edited by The Biochemical Society of Japan, “New Chemistry Experiment Course 18 @ Cell Culture Technology”, Tokyo Kagaku Dojin, 1990). Peptides are produced. As a medium that can be used for the culture, various types of commonly used media can be appropriately selected depending on the host cell used. For example, in the case of COS cells, for example, a medium in which a serum component such as fetal bovine serum (FBS) is added to a medium such as RPMI-1640 medium or Dulbecco's modified Eagle's minimum essential medium (DMEM) as necessary is used. can do. In the case of 293-EBNA cells, a medium obtained by adding G418 to a medium such as Dulbecco's modified Eagle minimum essential medium (DMEM) to which serum components such as fetal bovine serum (FBS) are added can be used.
By culturing the cell of the present invention, the polypeptide of the present invention that can be produced outside the cell of the cell can be obtained by various known separation procedures using the physical properties, biochemical properties and the like of the polypeptide (for example, , Edited by Masato Okada and Kaoru Miyazaki, "Revised Protein Experiment Note, Above and Below", Youtosha, 1999). Specifically, a culture solution containing the polypeptide of the present invention is treated with, for example, a normal protein precipitant, ultrafiltration, various liquid chromatography [eg, molecular sieve chromatography (gel filtration), adsorption chromatography, The polypeptide of the present invention can be purified by ion exchange chromatography, affinity chromatography, high performance liquid chromatography (HPLC), or the like], or dialysis, or a combination thereof.
The expression of the polypeptide of the present invention can be easily confirmed or purified by fusing the polypeptide of the present invention with the marker sequence in frame. Examples of the marker sequence include a FLAG tag, a hexa-histidine tag, a hemagglutinin tag, and a myc epitope. Further, by inserting a specific amino acid sequence recognized by a protease (for example, enterokinase, factor Xa, or thrombin) between the marker sequence and the polypeptide of the present invention, the marker sequence portion can be replaced with these proteases. Can be cut and removed.
[4] Detection method and screening method of the present invention
Using the polypeptide of the present invention, it is possible to detect whether or not a test compound inhibits the protease activity of the polypeptide of the present invention. Further, by using the detection method of the present invention, it is possible to screen for a substance that inhibits the protease activity of the polypeptide of the present invention. Since the polypeptide of the present invention, MDTS9 (Metalloprotease and Disintegran with with Thrombospondin type-1 repeats-9), is considered to be an ADAMTS protease from its sequence, it is considered to be a protease involved in extracellular matrix metabolism. As shown in Examples 5 and 8 described below, it is a protein expressed in the kidney, and as shown in Example 6 described below, it is an ADAMTS protease induced by TGF-β. Therefore, the substance that inhibits the protease activity of the polypeptide of the present invention has a high possibility of suppressing or inhibiting only the portion related to the qualitative change and the quantitative increase of the extracellular matrix component in the physiological action of TGF-β. Useful as an active ingredient of a therapeutic agent for chronic renal failure which is expected to be administered for a long period of time, the polypeptide of the present invention itself, a substance inhibiting the protease activity of the polypeptide of the present invention, or a substance for treating chronic renal failure Can be used as a screening tool.
The test compound that can be subjected to the detection method or the screening method of the present invention is not particularly limited. For example, various known compounds (including peptides) registered in a chemical file, combinatorial chemistry techniques (Terrett, NK, et al., Tetrahedron, 51, 8135-8137, 1995) or a group of compounds obtained by conventional synthetic techniques, or the phage display method (Felici, F. et al., J. Mol. Biol. , 222, 301-310, 1991) can be used. The known compounds include, for example, compounds (including peptides) which are known to have protease inhibitory activity, but it is not known whether or not the polypeptide of the present invention inhibits the protease activity. In addition, culture supernatants of microorganisms, natural components derived from plants or marine organisms, or animal tissue extracts can also be used as test compounds for screening. Furthermore, a compound (including a peptide) obtained by chemically or biologically modifying a compound (including a peptide) selected by the screening method of the present invention can be used.
The detection method of the present invention,
(1) a polypeptide of the present invention and (2) α₂Contacting macroglobulin with (3) a test compound, and
Polypeptide of the present invention and α₂Analyzing whether or not macroglobulin forms a complex that does not dissociate with SDS and / or a reducing agent (eg, 2-ME)
including.
In the detection method of the present invention, the test polypeptide and α₂-Instead of contacting with macroglobulin, the polypeptide of the invention and α₂-Can be carried out in the same manner as in the above-mentioned method for determining protease activity, except that macroglobulin is brought into contact with a test compound. That is, in the detection method of the present invention, the polypeptide of the present invention, the polypeptide for a substrate, and a test compound are contacted, and in the presence of the test compound, the polypeptide of the present invention and α₂The test compound inhibits the protease activity of the polypeptide of the invention by analyzing whether or not it forms a complex with macroglobulin that does not dissociate with SDS and / or a reducing agent (eg 2-ME) Detect whether to do. In the presence of a test compound, a polypeptide of the invention and α₂If the macroglobulin does not form a complex that does not dissociate with SDS and / or a reducing agent (eg, 2-ME), or if the degree of formation is reduced, the test compound may It can be determined that the protease activity of the peptide is inhibited.
In the screening method of the present invention, the detection method of the present invention detects whether or not a test compound inhibits the protease activity of the polypeptide of the present invention. Based on the result, the protease activity of the polypeptide of the present invention is determined. Or a substance for treating chronic renal failure. More specifically, for example, the polypeptide of the present invention and α₂Contacting a macroglobulin with a test compound, the polypeptide of the present invention and α in the presence of the test compound;₂-A substance that inhibits the protease activity of the polypeptide of the present invention or a substance for treating chronic renal failure can be selected by using the presence or absence or the degree of formation of a complex with macroglobulin as an index. In the presence of a test compound, a polypeptide of the invention and α₂If the macroglobulin does not form a complex that does not dissociate with SDS and / or a reducing agent (eg, 2-ME), or if the degree of formation is reduced, the test compound may The substance can be determined to be a substance that inhibits the protease activity of the peptide or a substance for treating chronic renal failure.
[5] Method for producing pharmaceutical composition for treating chronic renal failure of the present invention
The present invention includes a pharmaceutical composition for treating chronic renal failure, which comprises a substance that inhibits the protease activity of the polypeptide of the present invention, which is selected by the screening method of the present invention, as an active ingredient.
In a confirmation test of a quality standard of a pharmaceutical composition for treating chronic renal failure, the detection method of the present invention includes a step of detecting whether or not to inhibit the protease activity of the polypeptide of the present invention, and a formulation step, The present invention also includes a method for producing a pharmaceutical composition for treating renal failure.
Also, the present invention includes a method for producing a pharmaceutical composition for treating chronic renal failure, which comprises formulating a substance obtained by the screening method of the present invention including the detection step.
Formulations containing a substance that inhibits the protease activity of the polypeptide of the present invention as an active ingredient may be, depending on the type of the active ingredient, carriers, excipients, and / or other additives usually used for their formulation. Can be prepared.
Administration is, for example, oral administration such as tablets, pills, capsules, granules, fine granules, powders, or liquids for oral use, or injections such as intravenous injection or intramuscular injection, suppositories, transdermal administration Or parenteral administration by a transmucosal agent or the like. In particular, for peptides that are digested in the stomach, pharmaceutical preparations that do not undergo parenteral administration such as intravenous injection or digestion, for example, pharmaceutical preparations described in WO95 / 28963 are preferable.
In solid compositions for oral administration, one or more active substances and at least one inert diluent such as lactose, mannitol, glucose, microcrystalline cellulose, hydroxypropylcellulose, starch, polyvinylpyrrolidone, Alternatively, it can be mixed with magnesium metasilicate aluminate. The composition may contain additives other than an inert diluent, for example, a lubricant, a disintegrant, a stabilizer, or a solubilizing or solubilizing agent, according to a conventional method. The tablets or pills can be coated with a sugar coating or a film of a gastric or enteric substance, if necessary.
Liquid compositions for oral administration can include, for example, emulsions, solutions, suspensions, syrups, or elixirs; commonly used inert diluents, such as purified water or Ethanol can be included. The composition can contain additives other than inert diluents, for example, wetting agents, suspending agents, sweetening agents, flavoring agents, or preservatives.
Parenteral injections can include sterile aqueous or non-aqueous solutions, suspensions, or emulsions. The aqueous solution or suspension may contain, for example, distilled water for injection or physiological saline as a diluent. Examples of the diluent of the water-insoluble solution or suspension include propylene glycol, polyethylene glycol, vegetable oil (eg, olive oil), alcohols (eg, ethanol), polysorbate 80, and the like. The composition may further include a wetting agent, an emulsifying agent, a dispersing agent, a stabilizing agent, a solubilizing or solubilizing agent, or a preservative. The composition can be sterilized by, for example, filtration through a bacteria-retaining filter, blending of a bactericide, or irradiation. In addition, a sterile solid composition can be produced and dissolved in sterile water or another sterile injectable medium before use.
The dose can be appropriately determined in consideration of the strength of the activity of the active ingredient selected by the screening method, symptoms, age, sex, and the like of the administration subject.
For example, in the case of oral administration, the dose is usually about 0.01 to 1000 mg, preferably 0.01 to 100 mg per day for an adult (with a body weight of 60 kg). In the case of parenteral administration, the dosage is 0.01 to 1000 mg, preferably 0.01 to 100 mg per day in the form of injection.
[6] The antibody of the present invention or a fragment thereof
Antibodies that react with the polypeptide of the present invention (for example, polyclonal antibodies or monoclonal antibodies) can be obtained by directly administering the polypeptide of the present invention or a fragment thereof to various animals. Further, a DNA vaccine method (Raz, E., et al., Proc. Natl. Acad. Sci. USA, 91, 9519-9523, 1994; or Donnelly) was carried out using a plasmid into which a polynucleotide encoding the polypeptide of the present invention was introduced. JJ et al., J. Infect. Dis., 173, 314-320, 1996).
The polyclonal antibody is, for example, an animal immunized with an emulsion obtained by emulsifying the polypeptide of the present invention or a fragment thereof in an appropriate adjuvant (for example, Freund's complete adjuvant) intraperitoneally, subcutaneously, or vein. (Eg, rabbits, rats, goats, chickens, etc.) or eggs. A polyclonal antibody can be separated and purified from the thus-produced serum or egg by a conventional polypeptide isolation and purification method. Examples of such separation and purification methods include centrifugation, dialysis, salting out with ammonium sulfate, and chromatography using DEAE-cellulose, hydroxyapatite, protein A agarose, or the like.
Monoclonal antibodies can be easily produced by those skilled in the art by, for example, the cell fusion method of Koehler and Milstein (Kohler, G. and Milstein, C., Nature, 256, 495-497, 1975).
In other words, the polypeptide of the present invention or a fragment thereof is emulsified in an appropriate adjuvant (for example, Freund's complete adjuvant) repeatedly inoculated intraperitoneally, subcutaneously, or intravenously several times into mice every few weeks. Immunize with After the final immunization, the spleen cells are removed and fused with myeloma cells to produce a hybridoma.
As a myeloma cell for obtaining a hybridoma, a myeloma cell having a marker such as hypoxanthine-guanine-phosphoribosyltransferase deficiency or thymidine kinase deficiency (for example, mouse myeloma cell line P3X63Ag8.U1) can be used. . Further, as the fusogenic agent, for example, polyethylene glycol can be used. Further, as a medium for producing a hybridoma, for example, 10 to 30% of fetal bovine serum is appropriately added to a commonly used medium such as Eagle's minimum essential medium, Dulbecco's modified minimum essential medium, or RPMI-1640. In addition, it can be used. Fusion strains can be selected by the HAT selection method. Hybridoma screening is performed by using well-known methods such as ELISA method or immunohistochemical staining method using the culture supernatant, and a hybridoma clone secreting the desired antibody can be selected. Further, by repeating subcloning by the limiting dilution method, it is possible to guarantee the monoclonality of the hybridoma. The hybridoma thus obtained produces a purifiable amount of antibody by culturing it in the medium for 2 to 4 days or in the abdominal cavity of BALB / c mouse pretreated with pristane for 10 to 20 days. can do.
The monoclonal antibody thus produced can be separated and purified from the culture supernatant or ascites by a conventional polypeptide isolation and purification method. Examples of such separation and purification methods include centrifugation, dialysis, salting out with ammonium sulfate, and chromatography using DEAE-cellulose, hydroxyapatite, protein A agarose, or the like.
In addition, the antibody fragment containing the monoclonal antibody or a part thereof is obtained by incorporating all or a part of the gene encoding the monoclonal antibody into an expression vector and introducing the gene into an appropriate host cell (eg, Escherichia coli, yeast, or animal cell). It can also be produced.
The antibodies (including polyclonal antibodies and monoclonal antibodies) separated and purified as described above are digested with a polypeptide-degrading enzyme (for example, pepsin or papain) by a conventional method, and subsequently, the polypeptide is isolated by a conventional method. An antibody fragment containing a part of an active antibody, for example, F (ab ')₂, Fab, Fab ', or Fv.
Furthermore, an antibody reactive with the polypeptide of the present invention can be prepared by the method of Clackson et al. Or the method of Zebede et al. (Clackson, T. et al., Nature, 352, 624-628, 1991; or Zebedee, S. et al., Proc. Natl. Acad. Sci. USA, 89, 3175-3179, 1992) can be obtained as a single chain Fv or Fab. It is also possible to obtain a human antibody by immunizing a transgenic mouse (Lonberg, N. et al., Nature, 368, 856-859, 1994) in which the mouse antibody gene is replaced with a human antibody gene.
Example
Hereinafter, the present invention will be described specifically with reference to Examples, but these do not limit the scope of the present invention. In addition, unless otherwise indicated, it carried out according to a well-known method (for example, Sambrook, J., et al., "Molecular Cloning-A Laboratory Manual", Cold Spring Harbor Laboratory, NY, 1989).
Example 1: Preparation of C-terminal FLAG addition type expression vector
Plasmid pCEP4 (manufactured by Invitrogen) was cut with restriction enzymes ClaI and NsiI, blunt-ended, and then self-ligated to prepare an expression vector pCEP4d from which the EBNA1 expression unit derived from Epstein-Barr virus was removed. . After cutting the obtained expression vector pCEP4d with restriction enzymes NheI and BamHI, an agarose gel-extracted DNA fragment of about 7.7 kbp was added to an oligonucleotide having the nucleotide sequence represented by SEQ ID NO: 3. An expression vector pCEP4d-FLAG was prepared by inserting a double-stranded oligonucleotide obtained by annealing the oligonucleotide having the nucleotide sequence represented by SEQ ID NO: 4. The nucleotide sequence confirmed that the obtained expression vector had the target sequence.
Using the resulting expression vector pCEP4d-FLAG as a template, a combination of an oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 5 and an oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 6 as a primer was used as a pyrovest (PyroBest).^TM) PCR was performed using DNA polymerase (Takara Shuzo). In the PCR, a cycle consisting of 94 ° C. (30 seconds), 55 ° C. (30 seconds), and 72 ° C. (30 seconds) is repeated 15 times after first performing thermal denaturation at 94 ° C. (2 minutes). An extension reaction was performed at 72 ° C. (7 minutes). After the DNA fragment of about 0.4 kbp generated by the PCR is digested with the restriction enzyme SpeI, the DNA fragment is inserted into pCEP4d-FLAG (about 7.7 Kbp) digested with the restriction enzyme XbaI, thereby obtaining the expression vector pCEPdE2. -FLAG was created. In the obtained expression vector pCEPdE2-FLAG, the XbaI recognition sequence, the NheI recognition sequence, the NotI recognition sequence, the BamHI recognition sequence, and the FLAG tag, which are cloning sites, are arranged in this order downstream from the promoter.
Example 2: Cloning of full-length ORF gene of novel protease gene MDTS9
An oligonucleotide consisting of the base sequence represented by SEQ ID NO: 7 (a SpeI recognition sequence and a Kozak sequence are added to the 5 ′ side) and an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 8 (NotI: A human fetal kidney cDNA (Marathon-Ready)^TMDNA polymerase (TaKaRa LA Taq) using cDNA (manufactured by Clontech) as a template.^TMPCR was performed using Takara Shuzo Co., Ltd.). In the PCR, a cycle consisting of 98 ° C. (10 seconds) and 68 ° C. (2 minutes 30 seconds) is repeated 40 times after heat denaturation at 94 ° C. (2 minutes), and finally at 68 ° C. (7 minutes). Min) extension reaction. A DNA fragment of about 2.2 kbp (a SpeI recognition sequence and a Kozak sequence were added to the 5 ′ side and a NotI recognition sequence was added to the 3 ′ side) generated by the PCR was converted to a plasmid PCR2.1 (manufactured by Invitrogen). ) To obtain clone pMDTS9Cys1.
The obtained plasmid pMDTS9Cys1 was digested with restriction enzymes SpeI and NotI, and a DNA fragment of about 2.2 kbp was inserted into the XbaI site and NotI site of pCEPdE2-FLAG constructed in Example 1 to obtain plasmid pCEPdE2. -MDTS9Cys1-FLAG was constructed.
A combination of an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 9 (with a SpeI recognition sequence and a Kozak sequence added to the 5 ′ side) and an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 10 was used as a primer. , Human fetal kidney cDNA (Marathon-Ready)^TMDNA polymerase (TaKaRa LA Taq) using cDNA (manufactured by Clontech) as a template.^TMPCR was performed using Takara Shuzo Co., Ltd.). In the PCR, after first performing thermal denaturation at 94 ° C. (2 minutes), a cycle consisting of 98 ° C. (10 seconds) and 68 ° C. (30 seconds) is repeated 45 times, and finally 68 ° C. (7 minutes) Was performed. A DNA fragment of about 0.2 kbp (with a SpeI recognition sequence and a Kozak sequence added to the 5 ′ side) generated by the PCR was subcloned into a plasmid PCR2.1 (manufactured by Invitrogen) to obtain a clone pMDTS9 ( 5S2-12) was obtained.
The obtained plasmid pMDTS9 (5S2-12) was digested with restriction enzymes SpeI and NcoI to generate an approximately 0.2 kbp SpeI-NcoI DNA fragment A, and the previously obtained plasmid pMDTS9Cys1 was digested with restriction enzymes NcoI and NotI. The thus-produced NcoI-NotIΔDNA fragment B of about 2.0 kbp was inserted into the XbaI and NotI sites of pCEPdE2-FLAG constructed in Example 1 to construct a plasmid pCEPdE2-MDTS9Cys2-FLAG. Similarly, the plasmid pZErO-MDTS9Cys2 was constructed by inserting the DNA fragment A and the DNA fragment B into the SpeI and NotI sites of the plasmid pZErO-2 (Invitrogen).
Using, as a primer, a combination of the oligonucleotide having the nucleotide sequence represented by SEQ ID NO: 11 and the oligonucleotide having the nucleotide sequence represented by SEQ ID NO: 12, a human fetal kidney cDNA (Marathon-Ready)^TMDNA polymerase (TaKaRa LA Taq) using cDNA (manufactured by Clontech) as a template.^TMPCR was performed using Takara Shuzo Co., Ltd.). In the PCR, a cycle consisting of 98 ° C. (10 seconds) and 68 ° C. (2 minutes 30 seconds) is repeated 40 times after heat denaturation at 94 ° C. (2 minutes), and finally at 68 ° C. (7 minutes). Min) extension reaction. The DNA fragment of about 2.1 kbp generated by the PCR was subcloned into plasmid PCR2.1 (manufactured by Invitrogen) to obtain clone pMDTS9-3H.
A DNA fragment of about 2.1 kbp produced by cutting the obtained plasmid pMDTS9-3H with restriction enzymes SphI and NotI and the plasmid pCEPdE2-MDTS9Cys2-FLAG obtained by cutting the obtained plasmid pCEPdE2-MDTS9Cys2-FLAG with restriction enzymes SphI and NotI. The resulting plasmid was ligated with the approximately 9.3 kbp DNA fragment to construct a plasmid pCEPdE2-MDTS9Full-FLAG.
This plasmid pCEPdE2-MDTS9 Full-FLAG contains a gene consisting of the 1st to 3672nd bases in the nucleotide sequence represented by SEQ ID NO: 1 of the novel protease gene MDTS9, and has the amino acid sequence represented by SEQ ID NO: 2 A polypeptide having the amino acid sequence represented by SEQ ID NO: 21 added to the C-terminal of the sequence consisting of the 1st to 1224th amino acids can be expressed using animal cells as a host.
In addition, the plasmid pCEPdE2-MDTS9Cys2-FLAG obtained above contains a gene consisting of the 1st to 2250th nucleotides in the nucleotide sequence represented by SEQ ID NO: 1 of the novel protease gene MDTS9. A polypeptide having the amino acid sequence represented by SEQ ID NO: 21 added to the C-terminal of the sequence consisting of the 1st to 750th amino acids in the represented amino acid sequence can be expressed using an animal cell as a host. . The polypeptide having the amino acid sequence represented by SEQ ID NO: 2 is considered to be ADAMTS protease.
Example 3: Expression of MDTS9 short length protein (MDTS9Cys2) and MDTS9 full length protein (MDTS9Full)
Plasmid pCEPdE2-MDTS9Cys2-FLAG or plasmid pCEPdE2-MDTS9Full-FLAG prepared in Example 2 (or, as a control, plasmid pCEPdE2-FLAG prepared in Example 1) was used as a commercial transfection reagent (FuGENE).^TMSerum medium [DMEM (GIBCO-BRL), 10% fetal calf serum, 100 µg / mL penicillin, 100 µg / mL streptomycin, and 250 µg / mL using 6 Transfection Reagent (manufactured by Boehringer Mannheim) according to the attached instructions. -G418 (manufactured by Nacalai Tesque)]] into HEK293-EBNA cells (manufactured by Invitrogen).
After the introduction of the plasmid, the cells are cultured for 48 hours (hereinafter referred to as serum culture). Alternatively, after the introduction of the plasmid, the cells are cultured for 16 hours, washed twice with PBS, and then subjected to a serum-free medium [DMEM (GIBCO-BRL). ), 100 μg / mL penicillin, 100 μg / mL streptomycin, 250 μg / mL-G418 (manufactured by Nacalai Tesque)] for 32 hours (hereinafter referred to as serum-free culture).
Each culture solution obtained by the serum culture or the serum-free culture was centrifuged (3000 rpm, 10 minutes) using a centrifuge (Model 8800; manufactured by Kubota Seisakusho) to obtain a culture supernatant. Each cell remaining after removing the culture solution was extracted with an extract [20 mmol / L-HEPES (pH 7.4), 1% Triton X-100, 1% glycerol, 0.1% bovine serum albumin (BSA). ], The cells are detached from the culture plate by pipetting, and the obtained cell suspension is centrifuged (3000 rpm, 10 minutes) using a centrifuge (Model 8800; manufactured by Kubota Seisakusho). Thereby, it was fractionated into a cell membrane-bound fraction (supernatant) and a cell fraction (precipitate).
The expression of the target protein in each of the obtained fractions (that is, the culture supernatant, the cell membrane-bound fraction, and the cell fraction) was determined by an antibody against a FLAG tag added to the C-terminus (mouse anti-FLAG monoclonal antibody M2; manufactured by Sigma) ) Was confirmed by Western blotting. That is, each of the above fractions was subjected to electrophoresis using SDS / 10% to 20% acrylamide gel (manufactured by Daiichi Kagaku) under reducing conditions using 2-ME, and then subjected to poly blotting using a blotting apparatus. Transferred to vinylidene difluoride (PVDF) membrane. After blocking was performed by adding a blocking agent (Block Ace; manufactured by Dainippon Pharmaceutical Co., Ltd.) to the PVDF membrane after transfer, the mouse anti-FLAG monoclonal antibody M2 and a horseradish peroxidase-labeled rabbit anti-mouse IgG polyclonal antibody (Zymed) Or Tago Co., Ltd.). Alternatively, following the blocking, a biotinylated M2 antibody (manufactured by Sigma) and a horseradish peroxidase-labeled streptavidin (manufactured by Amersham Pharmacia Biotech) were sequentially reacted. After the reaction, the expression of the target protein was confirmed using a commercially available western blotting detection system (ECL western blotting detection system; manufactured by Amersham Pharmacia Biotech).
In SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of a protein (i.e., MDTS9 short-length protein) detected in each fraction obtained by serum-free culturing of cells into which plasmid pCEPdE2-MDTS9Cys2-FLAG was introduced. The apparent molecular mass was about 55-65 KDa in the culture supernatant, about 55-65 KDa in the cell membrane bound fraction, and 80-95 KDa in the cell fraction. On the other hand, proteins detected in each fraction obtained by serum-free culturing of cells into which the plasmid pCEPdE2-MDTS9 Full-FLAG was introduced (i.e., MDTS9 full-length protein) are mainly detected in the cell membrane-bound fraction and the cell fraction. The apparent molecular mass in SDS-PAGE was about 130 to 140 KDa.
Example 4: Confirmation of protease activity of MDTS9 short protein
(1) Construction of plasmid pCEPdE2-MDTS9Cys2E / Q-FLAG
Quick Change Site Directed Mutagenesis Kit (QuickChange^TMUsing Site-Directed Mutagenesis Kit (manufactured by Stratagene), Glu (glutamic acid) of His-Glu-Ser-Gly-His (SEQ ID NO: 22), which is considered to be the active center, to Gln (glutamine) according to the attached instructions. A plasmid pZErO-MDTS9Cys2E / Q containing the replaced gene MDTS9Cys2E / Q was prepared. In addition, as a template, the plasmid pZErO-MDTS9Cys2 prepared in Example 2 was used, and as a primer set, an oligonucleotide having the nucleotide sequence represented by SEQ ID NO: 13 and a nucleotide sequence represented by SEQ ID NO: 14 were used. Oligonucleotides were used.
The obtained plasmid pZErO-MDTS9Cys2E / Q is digested with restriction enzymes SpeI and NotI, and a DNA fragment of about 2.3 kbp is inserted into the XbaI and NotI sites of the plasmid pCEPdE2-FLAG constructed in Example 1 above. As a result, a plasmid pCEPdE2-MDTS9Cys2E / Q-FLAG was obtained.
(2) α₂-Confirmation of protease activity based on complex formation with macroglobulin
Plasmid pCEPdE2-MDTS9Cys2-FLAG prepared in Example 2 or plasmid pCEPdE2-MDTS9Cys2E / Q-FLAG prepared in Example 4 (1) (or plasmid pCEPdE2-FLAG prepared in Example 1 as a control) ) Was subjected to SDS-PAGE under reducing conditions using 2-ME and transferred to a PVDF membrane in the same manner as described in Example 3 above. A blocking agent (Block Ace; manufactured by Dainippon Pharmaceutical Co., Ltd.) was added to the PVDF membrane after the transfer to block the goat anti-α.₂-A macroglobulin antibody (manufactured by CEDARLANE) and a horseradish peroxidase-labeled rabbit anti-goat IgG polyclonal antibody (manufactured by Zymed Laboratories) were sequentially reacted. After the reaction, the expression of the target protein was confirmed using a commercially available western blotting detection system (ECL western blotting detection system; manufactured by Amersham Pharmacia Biotech).
In the culture supernatant of the serum culture derived from the cells transfected with the plasmid pCEPdE2-MDTS9Cys2-FLAG, it was detected in the culture supernatant of each serum culture derived from the cells transfected with the plasmid pCEPdE2-MDTS9Cys2E / Q-FLAG or the plasmid pCEPdE2-FLAG. An unapplied band of about 250 KDa was detected. This result indicates that the MDTS9 short protein (MDTS9Cys2)₂-A complex was formed with macroglobulin, thus confirming that MDTS9 short protein (MDTS9Cys2) has protease activity.
Example 5: Confirmation of tissue expression distribution of MDTS9 gene
A commercially available cDNA panel [Multiple Tissue cDNA (MTC manufactured by Clontech)^TM) Among the Panels, Human MTC Panel I (catalog number: K14220-1), Human MTC Panel II (catalog number: K1421-1), Human Fetal MTC Panel (catalog number: K1425-1), and Human Tumor MTC Panel ( Using the catalog number: K1422-1)], the tissue expression distribution of the MDTS9 gene was analyzed according to the following procedure.
That is, a DNA polymerase (TaKaRa @ LA @ Taq) was obtained by using a combination of an oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 15 and an oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 16 as a primer, and using the cDNA panel as a template.^TMPCR was performed using Takara Shuzo Co., Ltd.). In the PCR, first, thermal denaturation was performed at 94 ° C. (2 minutes), and then a cycle consisting of 98 ° C. (10 seconds) and 68 ° C. (1 minute 30 seconds) was repeated 44 times. Subsequently, the reaction solution was subjected to agarose gel electrophoresis, and a DNA fragment of about 1.1 kbp derived from the mRNA of the MDTS9 gene was detected. As a result, it was found that the mRNA of the MDTS9 gene was expressed in the kidney.
Example 6: Induction of MDTS9 gene expression by TGF-β
(1) Preparation of template cDNA
Normal human kidney proximal tubule epithelial cells [Clonetics] 5 × 10 6-well plate (Asahi Techno Glass)⁵And cultured for 1 day using a renal epithelial cell culture medium kit (manufactured by Kronetex). After converting to a serum-free renal epithelial cell medium and further culturing for one day, the medium was replaced with a serum-free renal epithelial cell medium to which TGF-β1 (manufactured by Sigma) was added to a final concentration of 10 ng / ml. Cultured for hours. The control group was replaced with a serum-free renal epithelial cell medium to which TGF-β1 was not added, and cultured for 24 hours.
Total RNA was prepared from each treatment group using a commercially available total RNA purification reagent (ISOGEN; manufactured by Nippon Gene). The obtained total RNA was reacted at 37 ° C. for 90 minutes using DNase (manufactured by Nippon Gene). 0.5 μg of the total RNA treated with DNase was converted to cDNA using Superscript First Strand System (for RT-PCR) (GIBCO-BRL).
(2) Quantification of mRNA of MDTS9 gene by quantitative PCR
The expression fluctuation analysis in normal human renal proximal tubular epithelial cells was performed using the cDNA prepared in Example 6 (1) as a template and a sequence detector (Prism7700 / Sequence / Detector; manufactured by Applied Biosystems). As the primer set, a combination of an oligonucleotide having the nucleotide sequence represented by SEQ ID NO: 17 and an oligonucleotide having the nucleotide sequence represented by SEQ ID NO: 18 was used. The PCR was performed using a commercially available PCR reagent (SYBR Green PCR core reagent; manufactured by Applied Biosystems), followed by an initial denaturation reaction at 95 ° C (10 minutes), followed by 94 ° C (15 seconds) and 60 ° C. (30 seconds) and a cycle reaction consisting of 72 ° C. (60 seconds) were repeated 40 times.
In addition, in order to calculate the gene expression level of human β-actin as an internal standard, the cDNA was used as a template, and was composed of an oligonucleotide having a base sequence represented by SEQ ID NO: 19 and a base sequence represented by SEQ ID NO: 20 Using the combination with the oligonucleotide as a primer set, PCR was performed under the same conditions. Further, in order to obtain a standard curve for calculating the mRNA expression level, the primer set (that is, the sequence of the primer set (that is, the sequence) was prepared using the TGF-β1 unstimulated human kidney proximal tubular epithelial cell cDNA prepared in Example 6 (1) as a template. A combination of an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 17 and an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 18, or an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 19 and SEQ ID NO: 20; PCR in combination with an oligonucleotide consisting of the represented base sequence) under the same conditions. In order to obtain the expression amount of the MDTS9 gene mRNA per a certain amount of total RNA, the expression amount of the MDTS9 gene mRNA under each condition was shown as a ratio to the β-actin gene expression amount under each condition. As a result, it was found that the mRNA expression of the MDTS9 gene mRNA was approximately 8-fold induced by TGF-β1.
Example 7: MDTS9 gene expression variation in rat renal failure model
(1) Preparation of template cDNA
cDNA was prepared from the kidney of a rat 5/6 nephrectomy model (Kenjiro Kimura, “Kidney and Dialysis”, 1991 extra edition, 431-439). After completion of 5/6 nephrectomy, 5/6 nephrectomized rats and sham-operated rats were each dissected at 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, and 10 weeks. Was immediately excised and immediately frozen and stored at -80 ° C. The kidneys of each group were crushed using a cell crusher (Cryopress CP-100; manufactured by Microtech Nichion) under liquid nitrogen freezing, and then totalized using a total RNA purification reagent (ISOGEN; manufactured by Nippon Gene). RNA was prepared. The extracted total RNA was reacted at 37 ° C. for 90 minutes using DNase (Nippon Gene). 0.25 μg of the total RNA treated with DNase was converted to cDNA by Superscript II First Strand System (for RT-PCR) (GIBCO-BRL).
(2) Quantification of mRNA of rat MDTS9 counterpart by quantitative PCR
The expression fluctuation analysis in the kidney in a rat renal failure model was performed using the cDNA prepared in Example 7 (1) as a template and a sequence detector (Prism 7700 \ Sequence \ Detector; manufactured by Applied Biosystems). An oligonucleotide consisting of the base sequence represented by SEQ ID NO: 23 and an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 24 were used as a primer set. The PCR was performed using a commercially available PCR reagent [Cyber Green PCR Core Reagent (manufactured by Applied Biosystems)], followed by an initial denaturation reaction at 95 ° C (10 minutes), followed by 94 ° C (15 minutes). ), 60 ° C (30 seconds) and 72 ° C (60 seconds) were repeated 45 times.
Further, in order to calculate the gene expression level of human glyceraldehyde 3-phosphate dehydrogenase (G3PDH) as an internal standard, an oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 25, PCR under the same conditions was performed using an oligonucleotide having the nucleotide sequence represented by No. 26 as a primer set. Furthermore, in order to obtain a standard curve used for calculating the mRNA expression amount, PCR was performed under the same conditions using rat genomic DNA (manufactured by Clontech) as a template and the primer set. In order to compare the expression level of rat MDTS9 gene mRNA with respect to a certain amount of total RNA in each group, the expression level of rat MDTS9 gene mRNA under each condition was shown as a ratio to the G3PDH gene expression amount under each condition. . As a result, in the 5/6 nephrectomy model, the rat MDTS9 gene mRNA was expressed about 5 times as much as the sham-operated rat one week after the operation, and the amount of urinary protein significantly increased after the operation. At 3 weeks, 6 weeks after the operation when the kidney weight began to increase remarkably compared with the normal kidney weight, and at 8 weeks after the operation when the disease state worsened, it was found that about twice as many genes were expressed, respectively.
This example revealed that the expression of the MDTS9 gene was induced in the renal failure model.
Example 8: Immunohistological staining of human kidney tissue section
(1) Preparation of anti-human MDTS9 antibody
First, plasmid pGEX-6P-1 (manufactured by Amersham Pharmacia Biotech) was prepared according to the experimental manual (edited by Masato Okada and Kaoru Miyazaki, “Revised Protein Experiment Note”, Yodosha, p. 162-179)]. Using an expression vector, a fusion protein (GST-MDTS9A) of a peptide consisting of amino acids 280 to 410 in the amino acid sequence represented by SEQ ID NO: 2 with glutathione S-transferase (GST) was used in Escherichia coli. To produce the inclusion body fraction. Subsequently, the inclusion body fraction was subjected to preparative SDS-polyacrylamide gel electrophoresis (PAGE), and the target GST-MDTS9A protein was extracted from the gel by a diffusion method (Masato Okada and Kaoru Miyazaki, edited protein revision). Experimental notebook ", Yodosha, pp. 48-51).
Rabbits (Japanese white species) were immunized with the obtained GST-MDTS9A protein 5 times at intervals of 10 to 14 days, and then an antiserum was prepared. From this antiserum, the IgG fraction was first affinity-purified on a Protein G Sepharose FF column (manufactured by Amersham Pharmacia Biotech), and then, from the amino acids 280 to 410 in the amino acid sequence represented by SEQ ID NO: 2. Affinity purification was performed on a column (MBP-MDTS9A column) on which a fusion protein (MBP-MDTS9A) of the resulting peptide and mannose-binding protein (MBP) was immobilized to obtain an anti-human MDTS9 antibody. Affinity purification on a Protein G Sepharose FF column, immobilization of MBP-MDTS9A on a CNBr-activated Sepharose FF column (manufactured by Amersham Pharmacia Biotech), and affinity purification on an MBP-MDTS9A column were performed according to the attached instructions. The production and purification of MBP-MDTS9A in E. coli was performed using pMAL-c2E (manufactured by New England Biolabs) as an expression vector and following the instructions of the company's "pMAL protein fusion and purification system".
(2) Detection of MDTS9 protein in human kidney
After reacting the anti-human MDTS9 antibody prepared in Example 8 (1) with formalin-fixed and paraffin-embedded tissue sections on a slide glass, a commercially available staining kit (Vectastain ABC-AP kit, catalog number AK- 5000; manufactured by Vector) according to the attached instructions. At that time, a biotin-labeled anti-rabbit antibody (catalog number BA-1000; manufactured by Vector) was used as a secondary antibody, and an alkaline phosphatase substrate kit I (catalog number SK-5100; manufactured by Vector) was used as a chromogenic substrate. As a result, staining of epithelial cells, particularly glomerular epithelial cells (podocytes), was observed in the kidneys of healthy individuals and diabetic nephropathy (early or late stage).
This example clearly shows that the MDTS9 protein is expressed in human kidney.
Industrial applicability
Since the polypeptide of the present invention is a novel protease expressed in kidney induced by TGF-β and involved in extracellular matrix metabolism, the substance that inhibits the protease activity of the polypeptide of the present invention is TGF-β. Among the physiological actions of -β, it is highly likely that only the portion related to qualitative change and quantitative increase of extracellular matrix components is suppressed or inhibited, and is useful as a therapeutic agent for chronic renal failure which is expected to be administered for a long time. That is, according to the polypeptide of the present invention, a simple screening system for a therapeutic agent for chronic renal failure can be provided. In addition, the polynucleotide, expression vector, cell, and antibody of the present invention are useful for producing the polypeptide of the present invention.
Sequence listing free text
The description of “Artificial @ Sequence” is described in the numerical heading <223> in the following sequence listing. Specifically, each base sequence represented by the sequences of SEQ ID NOs: 3 and 4 in the sequence listing is an artificially synthesized linker sequence. Each base sequence represented by the sequences of SEQ ID NOS: 5 to 9 and 12 to 14 in the sequence listing is a primer sequence artificially synthesized. Furthermore, the amino acid sequence represented by SEQ ID NO: 21 in the sequence listing is an amino acid sequence obtained by expression of DNA containing a nucleotide sequence encoding a restriction enzyme NotI recognition nucleotide sequence and a FLAG tag amino acid sequence.
As described above, the present invention has been described in connection with the specific embodiments. However, modifications and improvements obvious to those skilled in the art are included in the scope of the present invention.
[Sequence list]

Claims (15)

(1) a sequence consisting of the first to 750th amino acids in the amino acid sequence represented by SEQ ID NO: 2, and (2) a sequence consisting of the first to 750th amino acids in the amino acid sequence represented by SEQ ID NO: 2. In the sequence, the amino acid sequence has 1 to 10 amino acids deleted, substituted and / or inserted, or (3) consists of the 1st to 1224th amino acids in the amino acid sequence represented by SEQ ID NO: 2. A polypeptide comprising an amino acid sequence in which 1 to 10 amino acids have been deleted, substituted, and / or inserted in the sequence, and which exhibits protease activity. A polypeptide comprising a sequence consisting of the 1st to 750th amino acids in the amino acid sequence represented by SEQ ID NO: 2 and further exhibiting protease activity. (1) an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, and / or inserted in the sequence consisting of amino acids 1 to 750 in the amino acid sequence represented by SEQ ID NO: 2, or (2) in the sequence consisting of amino acids 1 to 1224 in the amino acid sequence represented by SEQ ID NO: 2, consisting of an amino acid sequence in which 1 to 10 amino acids have been deleted, substituted, and / or inserted; Moreover, a polypeptide exhibiting protease activity. (1) homology with the sequence consisting of the 1st to 750th amino acids in the amino acid sequence represented by SEQ ID NO: 2, or (2) the 1st to 1st amino acids in the amino acid sequence represented by SEQ ID NO: 2 A polypeptide comprising an amino acid sequence having 90% or more homology with the sequence consisting of the amino acid No. 1224, and showing protease activity. (1) homology with the sequence consisting of the 1st to 750th amino acids in the amino acid sequence represented by SEQ ID NO: 2, or (2) the 1st to 1st amino acids in the amino acid sequence represented by SEQ ID NO: 2 A polypeptide consisting of an amino acid sequence having 90% or more homology with the sequence consisting of the amino acid No. 1224, and showing protease activity. (1) consisting of the 1st to 750th amino acids in the amino acid sequence represented by SEQ ID NO: 2, or (3) starting from the 1st to 1224th amino acids in the amino acid sequence represented by SEQ ID NO: 2 Polypeptide. A polynucleotide encoding the polypeptide according to any one of claims 1 to 6. An expression vector comprising the polynucleotide according to claim 7. A cell transfected with the expression vector according to claim 8. An antibody or a fragment thereof that binds to the polypeptide according to any one of claims 1 to 6. The method for producing a polypeptide according to any one of claims 1 to 6, comprising culturing the cell according to claim 9 and recovering the polypeptide according to any one of claims 1 to 6. Method. (1) a polypeptide according to any one of claims 1 to 6, (2) alpha ₂ - and macroglobulin, (3) contacting a test compound, and the polypeptide and alpha ₂ - Macro A method for detecting whether or not a test compound inhibits the protease activity of the polypeptide, comprising a step of analyzing whether or not globulin forms a complex that does not dissociate with SDS and / or a reducing agent. A screening method for a substance that inhibits the protease activity of the polypeptide according to any one of claims 1 to 6, comprising a detection step by the method according to claim 12, and a step of selecting a substance that inhibits protease activity. Method. A method for screening a substance for treating chronic renal failure by the method according to claim 13. A method for producing a pharmaceutical composition for treating chronic renal failure, comprising a detection step by the method according to claim 12 and a formulation step.